EFFECTS OF IONIZING-RADIATION ON DEOXYRIBONUCLEIC-ACID .7. ELECTRON-CAPTURE AT CYTOSINE AND THYMINE

Exposure of a range of DNA samples in various media to Co-60 gamma-rays at 77 K gives electron-capture centres characterized by an EPR doublet with A(H-1) of ca. 16 G. Since the electron-adducts of C and T give very similar doublet EPR spectra in irradiated DNA, it is difficult to judge the proportions of C.- and T.- formation by inspection. The possibility, suggested by others, that computer fits can be used to give a quantitative measure of these species is discussed. However, in view of the variability of the features directly assignable to C.- and T.- units in different environments, we suggest that this approach has only qualitative significance. The alternative method involves annealing to convert T.- into TH. radicals in which a hydrogen atom is added to C6, the resulting radical having a completely characteristic octet EPR spectrum. It is argued that the ejected electrons move through the stacked DNA bases, becoming trapped at C or T depending upon the relative rates at which C.- and T.- are protonated to give C.-(H+) (protonated at N3) and T.-(H+) (protonated on oxygen). If this is correct, interconversion between C.- and T.- on annealing is unlikely, and only T.- can lead to TH. formation. This is also not accurate, since TH. decay sets in within the same temperature range as it is being formed. By generating TH. from frozen aqueous DNA with ultraviolet light, the pure decay annealing curve has been obtained, and using this we have been able to extrapolate the data for TH. from the gamma-irradiated samples to give the real yields of TH.. The results show that ca. 36% of the doublet must be due to T.- centres, the remainder (64%) being assigned to C.- centres. The ratio of C.- to T.- varies with the DNA source, and with the environment. We suggest that it is largely governed by the relative rates of protonation to give C.-(H+) and T.-(H+), and the factors controlling these rates are discussed. The use of lithium chloride glasses completely suppresses the formation of G.+ centres, leaving well-defined radical-anion spectra, but on annealing, conversion to TH. is negligible despite the rapid, and complete, loss of the doublet species. This result is discussed in terms of reaction with Cl2.- radicals formed in abundance in these glasses. Studies designed to detect any site-specificity in the DNA damage leading to strand breaks suggest that all possible sites are damaged. These results strongly support the postulate that yields of C.- and T.- are comparable. The possibility that some A.+ cations are formed in addition to G.+ cations is also considered in the light of these results.